Project Summary Ocular gene therapy has great potential for sustained expression of anti-angiogenic proteins for retinal/choroidal vascular diseases and also for replacement of mutant genes in patients with inherited retinal degenerations. Viral vectors provide efficient transduction of photoreceptors and retinal pigmented epithelial (RPE) cells when injected into the subretinal space, but not when injected into the vitreous cavity. Subretinal injections require vitrectomy, which carries small but significant risks of vision-threatening complications. Viral vectors induce an immune response which can cause bystander damage to retinal cells and potentially reduce the level and duration of transgene expression. Nonviral gene transfer is less immunogenic and also enables a large carrying capacity for genetic cargos, but has been limited by inefficient transfection. We have developed a new approach, suprachoroidal nonviral gene transfer, which provides widespread, prolonged, robust transgene expression in photoreceptors and RPE cells after suprachoroidal injection of engineered biodegradable polymeric nanoparticles. We propose to optimize the polymer structure and nanoparticle formulation, expression plasmid characteristics, and promoter to maximize the level, topographical extent, and duration of transgene expression in the retina and RPE cells. Suprachoroidal injections are performed in an outpatient clinic setting and can be safer than subretinal injections. The nanoparticles will be used for nonviral suprachoroidal gene transfer of vascular endothelial growth factor (VEGF)165, to determine if effects are comparable to those seen with high level expression of VEGF165 in transgenic mice and to establish new models of retinal/choroidal vascular disease in rats and rabbits. In parallel, we will create a new bioengineered protein that blocks angiogenesis by antagonizing a broad range of VEGF family ligands with high affinity and that is expressed at high levels by the newly engineered vector. After suprachoroidal injection of our new nonviral anti-VEGF vector, we will measure protein expression level, duration of expression, and therapeutic efficacy in established models of retinal/choroidal vascular disease and in the new disease models that we develop. These studies address a major unmet need in highly prevalent eye diseases by developing a noninvasive approach to achieve robust, long term suppression of VEGF family members. Further, the creation of an enabling nonviral gene delivery technology and a novel therapeutic anti-angiogenesis protein could be applicable to many other diseases as well.